Host-odour recognition in two tick species is coded in a blend of vertebrate volatiles

The questing behaviour of ixodid ticks serves for identification and localisation of approaching hosts and is evoked by carbon dioxide, vibrations, visual and odour stimuli. In an olfactometer, we examined the specificity of the questing response of larvae of Boophilus microplus, a one-host tick which develops mainly on cattle, and Ixodes ricinus, a three-host tick with a broader host spectrum. While all mammalian odours tested were equally stimulatory for I. ricinus, B. microplus was clearly more activated by bovine odours. A phenolic fraction of bovine odour stimulated B. microplus only. Attractive components of the host odours were identified by exposing the ticks to single chemicals and mixtures. Single chemicals stimulated questing responses only at levels higher than the levels detected in the bovine odour. However, an artificial odour blend of 37 pure chemicals, diluted to concentrations at which the individual components were inactive, proved to be as effective as natural host odour for both tick species. Further fractionation of the blend revealed that the combinatory effect was achieved by only 7 compounds in both species. Although B. microplus responded to the same synergistic mixture of volatiles as I. ricinus, it showed significant higher sensitivity to the cattle-associated compounds 1-octen-3-ol and 2-nitrophenol and this might contribute to its host-specificity. Accepted: 20 March 1999     

Expression of a synthetic protein-based polymer (elastomer) gene in Aspergillus nidulans

A gene for a synthetic protein-based polymer, G-(VPGVG)₁₁₉-VPGV, coding for the EG-120mer (elastomer), was cloned into a fungal expression vector to allow constitutive expression of the polymer controlled by the gpdA (glyceraldehyde-3-phosphate dehydrogenase) promoter sequence of Aspergillus nidulans. Stable transformants of A. nidulans showed plasmid integration with varying copy number when analyzed by Southern-blot hybridization. Expression of the synthetic gene was demonstrated by Northern-blot hybridization. However, the translational efficiency for production of the polymer polypeptide was low, presumably because of certain codons in the polymer gene (CCG and GUA) that are rarely used by A. nidulans. Partial purification by reversible phase transition followed by sodium dodecyl sulfate/polyacrylamide gel electrophoresis revealed the presence of polymer protein in a transformant that contained multiple copies of the polymer gene. This study represents the first attempt to express a synthetic gene (with no natural analog) in a fungus. Received: 23 July 1996 / Received revision: 19 November 1996 / Accepted: 23 November 1996     

Glucose diffusion in crosslinked proteins containing streptomyces

Summary Diffusion measurements of glucose in a crosslinked water-swollen protein matrix containingStreptomyces thermovulgaris strain 127, are described. Solute diffusion coefficients in the gel phase were found to be considerably lower than for free diffusion. The range of diffusion coefficients was 0.83×10⁻¹⁰ to 2.68×10⁻¹⁰m²/s at corresponding ratios to free diffusion values 0.11 to 0.38. It is shown that polymer structure does not pose any significant effect on mass transfer for the small glucose molecule.     

<Emphasis Type="Italic">Pichia pastoris</Emphasis> fermentation with mixed-feeds of glycerol and methanol: growth kinetics and production improvement

Fed-batch fermentation of a methanol utilization plus (Mut⁺) Pichia pastoris strain typically has a growth phase followed by a production phase (induction phase). In the growth phase glycerol is usually used as carbon for cell growth while in the production phase methanol serves as both inducer and carbon source for recombinant protein expression. Some researchers employed a mixed glycerol-methanol feeding strategy during the induction phase to improve production, but growth kinetics on glycerol and methanol and the interaction between them were not reported. The objective of this paper is to optimize the mixed feeding strategy based on growth kinetic studies using a Mut⁺ Pichia strain, which expresses the heavy-chain fragment C of botulinum neurotoxin serotype C [BoNT/C(Hc)] intracellularly, as a model system. Growth models on glycerol and methanol that describe the relationship between specific growth rate (μ) and specific glycerol/methanol consumption rate (ν _gly, ν _MeOH) were established. A mixed feeding strategy with desired μ _gly/μ _MeOH =1, 2, 3, 4 (desired μ _MeOH set at 0.015 h⁻¹) was employed to study growth interactions and their effect on production. The results show that the optimal desired μ _gly/μ _MeOH is around 2 for obtaining the highest BoNT/C(Hc) protein content in cells: about 3 mg/g wet cells. Electronic Publication     

Characterization of chemical defenses in ranid tadpoles against a fish predator

Tadpoles of some ranid species appear to possess chemical defenses against fish predators, but the chemicals have not been characterized. Here, we evaluated the vulnerability of three Japanese anuran tadpole species (Glandirana rugosa, Pelophylax nigromaculatus, and Hyla japonica) to a fish (Gnathopogon elongatus elongatus) and analyzed the defensive chemicals extracted from the unpalatable tadpoles. Additionally, we examined the defensive behavior of unpalatable tadpoles in response to fish chemical cues. The fish rejected both G. rugosa (83%) and P. nigromaculatus (48%), but not H. japonica (0%). Many of the rejected tadpoles survived (60–80%). Possible defensive chemicals were extracted by methanol from the skin of G. rugosa, but were not identifiable by gas chromatography–mass spectrometry because of small quantities. The chemicals have high polarity and non-volatility. When exposed to fish chemical cues, P. nigromaculatus decreased activity presumably as a defensive behavior, but G. rugosa did not. We demonstrated the presence of chemical defenses in at least two of these species and revealed that G. rugosa releases more effective or greater amounts of defense chemicals than P. nigromaculatus with respect to this fish predator. The increased efficacy of chemical defenses may correlate with decreasing defensive behavior.     

The effect of organochlorines and heavy metals on sex steroid-binding proteins in vitro in the plasma of nesting green turtles, Chelonia mydas

In this study on green turtles, Chelonia mydas, from Peninsular Malaysia, the effect of selected environmental toxicants was examined in vitro. Emphasis was placed on purported hormone-mimicking chemicals such as dichlorodiphenyltrichloroethane (DDT), dichlorodiphenyldichloroethylene, dieldrin, lead, zinc and copper. Five concentrations were used: high (1 mg/L), medium (10⁻¹ mg/L), low (10⁻² mg/L), very low (10⁻⁶ mg/L) and control (diluted carrier solvent but no toxicants). The results suggest that environmental pesticides and heavy metals may significantly alter the binding of steroids [i.e. testosterone (T) and oestradiol] to the plasma proteins in vitro. Competition studies showed that only Cu competed for binding sites with testosterone in the plasma collected from nesting C. mydas. Dieldrin and all heavy metals competed with oestradiol for binding sites. Furthermore, testosterone binding affinity was affected at various DDT concentrations and was hypothesised that DDT in vivo may act to inhibit steroid-protein interactions in nesting C. mydas. Although the precise molecular mechanism is yet to be described, DDT could have an effect upon the protein conformation thus affecting T binding (e.g. the T binding site on the steroid hormone binding protein molecule).     

Immobilization of lipase from Candida rugosa on a polymer support

Lipase from Candida rugosa was immobilized by adsorption onto a macroporous copolymer support. Under optimum conditions the maximum amount of protein bound was 15.4 mg/g and the immobilization efficiency was 62%. The kinetics of lipase binding to the selected polymer carrier was assessed by using a general model of topochemical reactions. The effect of temperature on adsorption was thoroughly investigated, as was the adsorption mechanism itself. Analysis of the proposed kinetic model and the specific kinetic parameters measured suggest that surface kinetics control the adsorption process. According to the activation energy (E _a) and the rate constant, k, the enzyme has rather a high affinity for the support's active sites. The immobilized enzyme was used to catalyse the hydrolysis of palm oil in a lecithin/isooctane reaction system, in which the enzyme's activity was 70% that of the free enzyme. Kinetic parameters such as maximum velocity (V _max) and the Michaelis constant (K _m) were determined for the free and the immobilized lipase. Following repeated use, the immobilized lipase retained 56% of its initial activity after the fifth hydrolysis cycle. Received: 3 April 1998 / Received revision: 28 July 1998 / Accepted: 29 July 1998     

Synergistic bactericidal activity of <Emphasis Type="Italic">Eremanthus erythropappus</Emphasis> oil or β-bisabolene with ampicillin against <Emphasis Type="Italic">Staphylococcus aureus</Emphasis>

The activity of Eremanthus erythropappus oil (EO) and some of its compounds and their potential synergistic interaction with ampicillin against different strains of Staphylococcus aureus were investigated. Determination of chemical composition of EO by gas chromatography-mass spectrometry (GC/MS) and bioguided chemical fractionation led to the identification of β-bisabolene as the main active compound. A synergistic bactericidal activity of EO or β-bisabolene with ampicillin against Staphylococcus aureus was observed in a time-kill assay. EO and β- bisabolene have the potential to restore the effectiveness of ampicillin against resistant S. aureus.     

Immunomodulative properties of conjugates composed of detoxified lipopolysaccharide and capsular polysaccharide of <Emphasis Type="Italic">Vibrio cholerae</Emphasis> O135 bound to BSA-protein carrier

O135 serotype Vibrio cholerae isolated from Slovak river was used as a source of surface polysaccharide antigens. Following detoxification procedure, fractions of polysaccharides were separated by size exclusion chromatography. Two resultant fractions were the capsular polysaccharide (M _w ∼ 197,000 Da) and the lipopolysaccharide fragment (M _w ∼ 13,300 Da). These materials were used for preparation of four novel glycoconjugates. Two of them containing detoxified lipopolysaccharide as antigen were prepared by original chemical method using the new biocompatible polymer as carrier of antigen. Additionally, other two conjugates were prepared by direct linking of capsular and detoxified lipopolysaccharide antigens to the protein carrier using adipic acid dihydrazide spacer. The immunogenicities (induced IgM, IgG, IgA antibodies) of all conjugates were determined by enzyme-linked immunosorbent assay. Polymer containing conjugates elicited higher levels of specific anti-lipopolysaccharide IgM and IgG antibodies in comparison with other conjugates without polymer carrier. Enhanced IgM vibriocidal activity of mice antisera was also evident here.     

Diet: tissue stable isotope fractionation of carbon and nitrogen in blood plasma and whole blood of male reindeer Rangifer tarandus

Estimates of diet derived from stable isotope analyses are sensitive to the accuracy of corrections made for diet-tissue fractionation. In particular, diet-tissue fractionation in reindeer Rangifer tarandus may be expected to differ significantly from the generic values often used in stable isotope dietary calculations, given the known values obtained from other ungulates and the complex digestive system and nutrient recycling characteristic of the species. We fed domestic reindeer a homogenous artificial diet of known isotopic value in order to directly determine diet-tissue isotopic fractionation of carbon and nitrogen, the main elements used in stable isotope dietary analyses. Diet-tissue fractionation values for blood plasma were +3.5 ± 0.1‰ (δ¹³C) and +4.2 ± 0.3‰ (δ¹⁵N). Diet-tissue fractionation values for whole blood were +3.7 ± 0.2‰ (δ¹³C) and +2.5 ± 0.3‰ (δ¹⁵N). Metabolic turnover rates were clearly sufficient for complete tissue replacement over the period of artificial feeding for blood plasma, but may not have been so for whole blood, especially for δ¹⁵N. Our values, except for whole blood δ¹⁵N, differ considerably from the generic values often used in dietary studies and interspecific comparisons of trophic niche. The results underline the importance of obtaining as specific as possible fractionation values for the species, tissue, and in some cases sex and physiological status of animals under examination, and the potential problems associated with assuming ‘generic’ fractionation values when comparing species, especially where digestive processes are dissimilar.     

Production of 2,3-butanediol from corncob molasses, a waste by-product in xylitol production

Corncob molasses, a waste by-product in xylitol production, contains high concentrations of mixed sugars. In the present study, corncob molasses was used to produce 2,3-butanediol (BD) using Klebsiella pneumoniae SDM. This was the first report on the use of corncob molasses to produce bulk chemicals. Our results indicated that K. pneumoniae SDM can utilize various sugars contained in the corncob molasses in a preferential manner: glucose > arabinose > xylose. It was shown that high sugars concentration had an inhibitory effect on the cells growth and BD production. The maximum concentration of BD was 78.9 g/l after 61 h of fed-batch fermentation, giving a BD productivity of 1.3 g/l h and a yield of 81.4%. The present study suggests that the low-cost corncob molasses could be used as an alternative substrate for the production of BD by K. pneumoniae SDM, as well as a potential carbon source for production of other high-value chemicals.     

Immobilization of β-glucosidase on Eupergit C for Lignocellulose Hydrolysis

β-Glucosidase is frequently used to supplement cellulase preparations for hydrolysis of cellulosic and lignocellulosic substrates in order to accelerate the conversion of cellobiose to glucose. Typically, commercial cellulase preparations are deficient in this enzyme and accumulation of cellobiose leads to product inhibition. This study evaluates the potential for recycling β-glucosidase by immobilization on a methacrylamide polymer carrier, Eupergit C. The immobilized β-glucosidase had improved stability at 65 °C, relative to the free enzyme, while the profile of activity versus pH was unchanged. Immobilization resulted in an increase in the apparent K_m from 1.1 to 11 mm and an increase in V_max from 296 to 2430 μmol mg⁻¹ min⁻¹. The effect of immobilized β-glucosidase on the hydrolysis of cellulosic and lignocellulosic substrates was comparable to that of the free enzyme when used at the same level of protein. Operational stability of the immobilized β-glucosidase was demonstrated during six rounds of lignocellulose hydrolysis. Received 22 August 2005; Revisions requested 20 September 2005; Revisions received 8 November 2005; Accepted 10 November 2005     

Acidity enhances the effectiveness of active chemical defensive secretions of sea hares, <Emphasis Type="Italic">Aplysia californica</Emphasis>, against spiny lobsters, <Emphasis Type="Italic">Panulirus interruptus</Emphasis>

Sea hares such as Aplysia californica, gastropod molluscs lacking a protective shell, can release a purple cloud of chemicals when vigorously attacked by predators. This active chemical defense is composed of two glandular secretions, ink and opaline, both of which contain an array of compounds. This secretion defends sea hares against predators such as California spiny lobsters Panulirus interruptus via multiple mechanisms, one of which is phagomimicry, in which secretions containing feeding chemicals attract and distract predators toward the secretion and away from the sea hare. We show here that ink and opaline are highly acidic, both having a pH of ∼5. We examined if the acidity of ink and opaline affects their phagomimetic properties. We tested behavioral and electrophysiological responses of chemoreceptor neurons in the olfactory and gustatory organs of P. interruptus, to ink and opaline of A. californica within their natural range of pH values, from ∼5 to 8. Both behavioral and electrophysiological responses to ink and opaline were enhanced at low pH, and low pH alone accounted for most of this effect. Our data suggest that acidity enhances the phagomimetic chemical defense of sea hares.     

Biodegradation of a phenolic mixture in a solid–liquid two-phase partitioning bioreactor

A solid–liquid two-phase partitioning bioreactor (TPPB) in which the non-aqueous phase consisted of polymer (HYTREL) beads was used to degrade a model mixture of phenols [phenol, o-cresol, and 4-chlorophenol (4CP)] by a microbial consortium. In one set of experiments, high concentrations (850 mg l⁻¹ of each of the three substrates) were reduced to sub-inhibitory levels within 45 min by the addition of the polymer beads, followed by inoculation and rapid (8 h) consumption of the total phenolics loading. In a second set of experiments, the beneficial effect of using polymer beads to launch a fermentation inhibited by high substrate concentrations was demonstrated by adding 1,300 and 2,000 mg l⁻¹ total substrates (equal concentrations of each phenolic) to a pre-inoculated bioreactor. At these levels, no cell growth and no degradation were observed; however, after adding polymer beads to the systems, the ensuing reduced substrate concentrations permitted complete destruction of the target molecules, demonstrating the essential role played by the polymer sequestering phase when applied to systems facing inhibitory substrate concentrations. In addition to establishing alternative modes of TPPB operation, the present work has demonstrated the differential partitioning of phenols in a mixture between the aqueous and polymeric phases. The polymeric phase was also observed to absorb a degradation intermediate (arising from the incomplete biodegradation of 4CP), which opens the possibility of using solid–liquid TPPBs during biosynthetic transformation to sequester metabolic byproducts.     

Host use of a specialist lichen-feeder: dealing with lichen secondary metabolites

Host use by herbivores is largely determined by host properties such as nutrient content and chemical defence against foragers. The impacts of these attributes on a herbivore may largely depend on its life cycle stage. Lichen species are known to differ in nutritional quality and level of chemical defence and, consequently, vary as fodder for herbivores. The aim of this study was to explore the impact of several lichen species and the presence of their secondary metabolites on their use as hosts by a specialist lichen-feeder, Cleorodes lichenaria. This study also addressed, for the first time, how a specialist lichen-feeder deals with different lichen secondary metabolites. In the beginning of their development, larvae grew better on Xanthoria parietina than on the other host lichens, whereas older larvae grew best on Ramalina fraxinea. Lichen secondary chemicals in R. fraxinea and Parmelia sulcata hindered larval growth in the beginning but after 75 days lichen secondary chemicals had no impact on the mass of larvae. Physodic acids in Hypogymnia physodes were lethal to larvae. In general, larvae metabolized 70–95% of ingested lichen secondary chemicals and the rest of these were excreted in frass. Lichen secondary metabolites in P. sulcata restrict and in H. physodes prevent their use as a host for C. lichenaria larvae. Our main finding, the ability of larvae to metabolize several lichen secondary metabolites, indicates digestive adaptation to these chemicals. No signs of sequestration of these chemicals were found.     

Generation Characteristics of Highly Uniform Nonspherical Droplets of Soybean Oil Using Microchannel Array Devices

This paper investigated the generation characteristics of nonspherical oil-in-water (O/W) droplets consisting of food-grade components using microchannel (MC) array devices that have many rectangular MCs with shallow wells. The well height was designed to be twice the MC height. Two hydrophilic MC array devices made of surface-oxidized single-crystal silicon with equivalent MC diameters of 3.2 and 8.4 μm were used. Refined soybean oil was used as the to-be-dispersed phase, and a Milli-Q water solution of 1.0 wt% polyoxyethylene (20) sorbitan monolaurate (Tween20) was used as the continuous phase. Highly uniform discoid droplets with diameters of 9.0 and 21.5 μm, heights of 4.6 and 9.8 μm, and coefficients of variation of less than 4% were generated by simply forcing a to-be-dispersed phase via rectangular MCs into a well filled with a continuous phase. The to-be-dispersed phase pressures necessary for droplet generation were less than 8 kPa. The detailed generation process of the discoid droplets was analyzed using movie clips taken by a high-speed camera. Key phenomena during the detachment process were considered to be rapid flow of the to-be-dispersed phase into the well and instantaneous pinch-off of the neck. The effect of the to-be-dispersed phase velocity inside a rectangular MC (U _d,MC) on the resultant droplet diameter and the droplet-generation rate was also analyzed. Size-controlled discoid droplets were stably generated via the rectangular MC below the critical U _d,MC, and the droplet-generation rate became maximum at the critical U _d,MC.     

Effect of Different Levels of Selenium Supplementation on Growth Rate,Nutrient Utilization,Blood Metabolic Profile,and Immune Response in Lambs

The nutritional role of silver for the freshwater crustacean, Daphnia magna, was examined through four generations of deprivation. Silver inclusion in animal media was set at a nominal zero (employing chemicals of the highest available purity). Both reproduction (−60%) and life span (−40%) were negatively affected when compared to animals reared in the presence of 0.4 ng g⁻¹ Ag. These results strongly suggest a nutritional requirement of silver for daphnids at nanomolar concentrations.     

Changes in Hg fractionation in soil induced by willow

This study investigated the effect of willow (Salix viminalis ×  S. schwerinii) on soil characteristics, including changes in Hg fractionation in the soil solid phase, and Hg accumulation and distribution in pot-grown plants cultivated for 32 and 76 days in aged Hg-contaminated soil (30 mg Hg kg⁻¹DW). Changes in soil pH and organic carbon content as well as in Hg fractionation were monitored in both rhizospheric soil and in soil without plants. Mercury fractionation was performed by a 5-step sequential soil extraction procedure. Organic carbon content increased while pH decreased in the rhizospheric soil. Both chemically defined exchangeable Hg (0.1) and Hg bound to humic and fulvic acids (1.1) decreased in the rhizospheric soil, whereas plant accumulation of Hg increased with cultivation time. The sum of the decrease of these two soil Hg fractions after 76 days of cultivation was approximately equal to the amount of Hg accumulated in plants. Furthermore, the major Hg fractions (Hg bound to residual organic matter (53), sulphides (43), and the residual fraction (2.5)) remained stable. Neither whole plant accumulation of Hg from the soil, approximately 0.2 of total Hg in soil after 76 days cultivation, nor the fraction of total plant Hg in the shoots, which accounted for about 3 of the total plant Hg pool regardless of the cultivation time, were high. The overall results suggest that plants might be suitable for phytostabilization of aged Hg-contaminated soil, where root systems trap bioavailable Hg and help to control both leaching of Hg and re-entrainment of Hg-containing particulates from a contaminated site.     

The effect of irradiance and redox-modifying reagents on the 52 kDa protein disulfide isomerase of <Emphasis Type="Italic">Arabidopsis</Emphasis> chloroplasts

Immunoblot analysis was used to assess the effects of light and redox-modifying chemicals on the 52 kDa protein disulfide isomerase (PDI) from chloroplasts of Arabidopsis thaliana. A monoclonal antiserum was used that preferentially cross-reacts with the 52 kDa relative to the 65 kDa isoform of PDI. The PDI-52 was most abundant in leaves, flowers, stems and seeds, but was undetected in roots. PDI-52 formed a ∼220 kDa protein complex on blue native gels, indicating that it associates with either itself or other proteins in chloroplasts. Light decreased the levels of PDI-52 by 80 %, relative to the control protein (the CF1 subunit of chloroplast ATP synthase). Treatment with dithiothreitol decreased the content of the 52 kDa protein by half. In contrast, when the reduction of plastoquinone is blocked by DCMU, or when the plants are treated with phosphate, PDI-52 contents increased by 1.5 to 2-fold relative to CF1. The effect of the chemical treatments coincided with the effect of the light/dark cycle and implied that light decreased PDI-52 protein content by way of the cellular redox environment.     

Scanning electron microscopy of polyhydroxyalkanoate degradation by bacteria

Bacterial degradation of sheets of selected polyhydroxyalkanoates by Comamonas sp., Pseudomonas lemoignei and Pseudomonas fluorescens GK13 is reported. Five natural polyhydroxyalkanoates were used, namely poly(3-hydroxybutyrate), poly(3-hydroxyvalerate), a copolymer of 3-hydroxybutyrate and 3-hydroxyvalerate, a copolymer of mainly 3-hydroxyoctanoate and minor amounts of 3-hydroxyhexanoate, and two rubber-like copolymers of saturated and unsaturated hydroxyalkanoic acids that had been modified by electron-beam-induced cross-linking. Each of these polymers was degraded by at least one bacterial strain, the rate of hydrolysis being dependent on the surface area of the polymer exposed to attack. Scanning electron microscopy of partially degraded samples showed that hydrolysis started at the surface and at physical lesions in the polymer and proceeded to the inner part of the material. No evidence for areas of non-degradable polymer was found for any of the polymers analysed, even if the polymer contained chemical cross-links. Received: 24 July 1996 / Accepted: 29 August 1996